The goal of our study is to determine the chemical structure of the outermost surface component of anthrax spores, exosporium polysaccharide (EPS). Spores of Bacillus anthracis are the causative agent of the highly lethal anthrax infection. The anthrax spore consists of several distinct structural layers, which protect the core genetic material. Because of their unique structural properties, anthrax spores are long-lived and extremely resistant to adverse environments. Once inside the host, the same structures allow anthrax spores to survive the host immune surveillance and to germinate, leading to rapidly replicating bacilli and production of toxins that kill the host. Despite these facts, we know surprisingly little about the basic chemical structures of anthrax spore constituents and their functions. EPS is expressed on the surface of anthrax spores, forming a hair-like cover of the spore. EPS remains intact throughout the entire germination stage from dormant spores to newly transformed vegetative bacteria. Because polysaccharides are typically highly stable biopolymers of poor immunogenicity, we hypothesize that EPS plays significant roles in protecting the survival of spores and in shielding spores from immune defense. The elucidation of the EPS structure will provide vital fundamental understanding of anthrax spore biology and more generally of sporulating microorganisms. The EPS structure will reveal useful antigens for the development of methods for rapid detection and efficient destruction of anthrax spores and/or unprecedented spore-directed anthrax vaccine and therapy. Aim 1. Isolation of EPS from B. anthracis spores. Spores will be prepared from attenuated, avirulent strains of B. anthracis. The exosporium layer will be extracted from the spores. EPS will be cleaved from exosporium proteins by biochemical and enzymatic methods. EPS will be purified by gel filtration and/or ion exchange chromatography. Aim 2. Elucidation of the chemical structure of EPS. A combination of multidimensional high-resolution NMR spectroscopy, mass spectrometry, biochemical or enzymatic degradation, chemical derivatization, and gas chromatography or HPLC will be employed in parallel to elucidate the chemical structure of EPS. If EPS is found to be heterogenous, we will attempt to characterize the structures of all distinct entities.